FR2847264A1 - New DNA encoding mutant form of LysE protein, useful for transformation of methanol-utilizing bacteria for production of lysine and arginine, also new transformants - Google Patents

Abstract

DNA (I) that encodes a mutant (II) of the LysE (lysine export) protein of a coryneform bacterium, or its homolog, is new. DNA (I) that encodes a mutant (II) of the LysE (lysine export) protein of a coryneform bacterium, or its homolog, is new. (II) is a 236 amino acid (aa) sequence (2), reproduced, in which at least Gly56 has been replaced by a different aa, optionally with one or more other aa substituted, deleted, inserted or added. When (I) is introduced into a methanol-utilizing bacterium it confers resistance to a lysine analog (III). Independent claims are also included for: (1) bacterium (A) of the genera Methylophilus or Methylobacillus into which (I) has been introduced, in expressible form, and which can produce L-Lys or L-Arg; and (2) producing L-Lys and L-Arg by culturing (A).

Description

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 The present invention relates to a microbial engineering technique useful for the production of amino acids and more specifically a DNA encoding a mutant LysE protein, a microorganism containing this DNA and a method for producing L-amino acids such as L-lysine or L-arginine by fermentation using this microorganism.

 L-amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine, L-isoleucine, L-valine and L-phenylalanine are industrially produced by fermentation using micro -organisms belonging for example to the genera Brevibacterium, Corynebacterium, Bacillus, Escherichia, Streptomyces, Pseudomonas, Arthrobacter, Serratia, Penicillium, Candida. Bacterial strains isolated from natural or artificial mutants of these bacterial strains are often used to improve the productivity of these microorganisms. In addition, various techniques have been described for increasing the production of L-amino acids from these strains by increasing the activity of L-amino acid biosynthetic enzymes by means of genetic engineering techniques.

 The production of L-amino acids has increased considerably by culturing microorganisms such as those mentioned above and because of corresponding improvements in the production processes.

However, in order to meet the increasing demand in the future, the development of processes for more efficient production of L-amino acids at lower cost is necessary.

 Methanol is a known fermentation raw material that is available in large quantities at low cost. Processes for producing L-amino acids by fermentation with methanol are known and include methods using microorganisms that belong to the genus Achromobacter or Pseudomonas (Japanese Patent Application Laid-Open (Kokoku) No. 45-25273), Protaminobacter (Application Japanese Patent Laid-open Patent Application (Kokai) No. 49-125590), Protaminobacter or Methanomonas (Japanese Patent Application Laid-Open (Kokai) No. 50-25790), Microcyclus (Japanese Patent Application Laid-Open available to the public (Kokai) No. 52-18886), Methylobacillus (Japanese Patent Application Laid-Open (Kokai) No. 4-91793), Bacillus (Japanese translation of PCT Application (Kohyo) No. 3-505284 (WO 90/12105).

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 Applicant has developed methods for producing L-amino acids by culturing bacteria of the genera Methylophilus and Methylobacillus using artificial mutagenesis and genetic engineering techniques (published international application WO 00/61723; Japanese patent application made available to the Public (Kokai) No. 2001-120269).

 In recent years, proteins have been identified whose function is to specifically secrete an L-amino acid outside a cell or a microorganism, as well as the genes encoding these proteins. In particular, Vrljic et al. have identified a gene involved in the extracellular secretion of L-lysine by a Corynebacterium bacterium (Molecular Microbiology 22: 815-826 (1996)). This gene has been termed lysE and it has been described that the ability to produce L-lysine of Corynebacterium bacteria could be improved by increasing the expression of this gene in bacteria (published international patent application WO 97/23597). . It is also known that the production of several types of L-amino acids can be improved by increasing the expressed amounts of amino acid-secreting proteins in Escherichia coli (Japanese Patent Application Laid-open No. 2000-189180). For example, it has been reported that the production of cystine and cysteine could be improved by increasing the expression of the ORF 306 gene in Escherichia coli (European Patent Application Laid-open No. 885962).

 However, improving the production of L-amino acids by improving their secretion by bacteria assimilating methanol during fermentation has not yet been described. In addition, an amino acid secretion gene that may exhibit secretory activity in bacteria assimilating methanol has not been described.

 Thus, it is an object of the present invention to provide a process for efficiently producing L-lysine or L-arginine with methanol, which is available in abundance and at low cost.

 The present invention also aims to provide a DNA encoding a mutant of the LysE protein, or a protein homologous thereto, of a coryneform bacterium where the mutant, or the homologous protein, confers resistance to an analogue of the L-lysine to a bacterium assimilating the methanol in which it is introduced.

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 The present invention also relates to such a DNA wherein the mutant is a protein defined as follows: (A) a protein that has the amino acid sequence of SEQ ID NO: 2, wherein at least the glycine residue at position 56 is replaced by another amino acid, or (B) a protein which has the amino acid sequence of SEQ ID NO: 2 where at least the glycine residue at position 56 is replaced by another amino acid residue, and one or more Amino acid residues at different positions of the 56th residue are substituted, deleted, inserted or added, and when said mutant is introduced into a methanol-assimilating bacterium it confers resistance to an L-lysine analogue.

 The present invention also relates to such a DNA wherein said DNA is selected from the group consisting of A) a DNA which has the nucleotide sequence of SEQ ID NO: 1 where a mutation leads to the replacement of at least the 56th residue (glycine) of the protein encoded by another amino acid residue, or B) a DNA which is hybridizable with the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, or a probe prepared from said nucleotide sequence, DNA or probe which, when it is introduced into a bacteria assimilating methanol, gives it resistance to an analogue of L-lysine.

 The present invention also relates to such a DNA wherein the other amino acid residue is a serine residue.

 The present invention also relates to said DNA wherein the L-lysine analogue is S- (2-aminoethyl) cysteine.

 The present invention also relates to said DNA wherein the bacterium assimilating methanol is a bacterium belonging to the genus Methylophilus or Methylobacillus.

 The present invention also relates to a bacterium belonging to the genus Methylophilus or Methylobacillus in which the above DNA is introduced in a form that can be expressed and is capable of producing L-lysine or L-arginine.

 The present invention also relates to a process for producing L-lysine or L-arginine comprising the steps of A) culturing the bacterium described above in a medium for producing and accumulating L-lysine or L-lysine. arginine in the medium, and B) the collection of L-lysine or L-arginine from the culture.

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 The present invention also relates to such a process in which the medium contains methanol as the main carbon source.

 According to the present invention, it is possible to improve the productivity of bacteria assimilating methanol with respect to L-amino acids, in particular with regard to L-lysine and L-arginine.

 The present invention will be better understood on reading the detailed description which follows and refers to the accompanying drawing, given solely by way of example and in which: FIG. 1 represents a map of the plasmid pRStac having the tac promoter and the plasmid pRSIysE which was constructed by insertion of the lysE gene into the plasmid pRStac.

 The applicant initially found that when an L-amino acid, in particular L-lysine or L-arginine, is produced by means of bacteria assimilating methanol, in particular bacteria belonging to the genera Methylophilus and Methylobacillus, the extracellular secretion of these L-amino acids did not occur. Then, the Applicant was able to successfully obtain genes which exhibited an activity of secretion of amino acids, especially in these microorganisms, and thus found that it was possible to efficiently produce amino acids using the genes thus obtained.

 The Applicant has introduced the already known lysis gene, which is derived from Corynebacterium bacteria, into a bacterium that assimilates methanol, and examined its effect on the production of amino acids. She found that the introduction of the lysis gene into a bacterium that assimilated methanol led to a mutation or deletion so that the lysE gene was not functional. Typically, secretory proteins must be incorporated into the cell membrane to be functional so that protein and membrane conditions such as lipid composition must be compatible with each other. It was concluded that it would be difficult to express a heterologous membrane protein, such as LysE, for the protein to be functional, and this conclusion was supported by the previously mentioned result.

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 Thus, the Applicant has found a mutant gene that was functional in a methanol-assimilating bacterium by searching for the L-amino acid secretion gene mentioned above. In addition, the Applicant has noted a marked effect in the use of this mutant gene in the production of amino acids by means of a bacteria assimilating methanol. The Applicant has successfully obtained a plurality of mutant genes that are functional in bacteria assimilating methanol.

 The DNA of the present invention encodes a mutant of the LysE protein, or a protein homologous to the LysE protein, derived from a coryneform bacterium, which may exhibit a function of the LysE protein when it is introduced into a bacterium assimilating methanol. .

 "LysE protein function" means at least one of the functions defined as follows: (1) a function conferring resistance to an L-lysine analogue when expressed in a methanol-assimilating bacterium introducing the DNA encoding the mutant mentioned above.

 The term "conferring resistance to an L-lysine analogue" to the bacterium means that upon introduction of the DNA encoding the LysE mutant into the aforementioned methanol-assimilating bacteria, the bacterium is able to grow in the presence of a higher concentration of L-lysine analogue than in the case of bacteria that do not contain this DNA, for example wild-type strains of bacteria assimilating methanol. For example, after culturing on an agar medium containing an L-lysine analogue at a certain concentration for a certain duration, if a transforming strain of the bacterium assimilating the methanol in which said DNA is introduced forms colonies, while a strain Since the non-transformant does not form colonies, it can be concluded that resistance to the L-lysine analogue has been conferred on said transformant strain. The analogue of L-lysine can be, for example, S- (2-aminoethyl) cysteine.

 (2) a function of improving the extracellular secretion of L-lysine and / or L-arginine, when said mutant is introduced into a bacteria assimilating methanol.

 The expression "enhancement of the extracellular secretion of L-lysine and / or L-arginine" means that when a bacterium assimilating

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 the methanol containing the DNA according to the present invention is cultured, the amount of L-lysine and / or L-arginine secreted in the medium is increased relative to the bacteria assimilating methanol which does not contain the DNA of the present invention invention. Improving the extracellular secretion of L-amino acids results in an increased concentration of the L-amino acid accumulated in the medium during the cultivation of a bacterium assimilating the methanol containing the DNA of the present invention, compared to a bacterium assimilating methanol that does not contain this DNA, as a result of the introduction of DNA. In addition, enhancement of extracellular secretion of L-amino acid can also be observed when a lower concentration of intracellular L-amino acid is detected upon introduction of the DNA of the present invention into a methanol-assimilating bacterium.

comme Methylobacillus glycogenes et Methylobacillus f7agellatum. According to the present invention, the bacterium assimilating methanol is a bacterium that can grow using methanol as the main carbon source, and in which the function of the LysE protein is expressed when the DNA according to the present invention is introduced. Such a bacterium may for example be a Methylophilus bacterium such as a bacterium Methylophilus methylotrophus or Methylobacillus

such as Methylobacillus glycogenes and Methylobacillus f7agellatum.

 The strain AS1 (NCIMB10515) is an example of the bacterium Methylophilus methylotrophus. Methylophilus methylotrophus strain AS1 (NCIMB10515) is available from National Collections of Industrial and Marine Bacteria (Address: NCIMB Lts., Torry Research Station, 135 Abbey Road, Aberdeen AB9 8DG, United Kingdom).

 The Methylobacillus glycogenes bacteria include, but are not limited to, strain T-11 (NCIMB11375), ATCC strain 21276, ATCC strain 21371, ATR80 strain (described in Appl Microbiol.

Biotechnol., 42, p. 67-72 (1994)), strain A513 (described in Appl., Microbiol Biotechnol., 42, pp. 67-72 (1994)). The strain Methylobacillus glycogenes NCIMB 11375 is available from the National Collections of Industrial and Marine Bacteria (Address: NCIMB Lts., Torry Research Station, 135 Abbey Road, Aberdeen AB9 8DG, United Kingdom). Strain KT is an example of the bacterium Methylobacillus flagellatum (described in Arch Microbiol., 149, pp. 441-446 (1988)).

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 One embodiment of the DNA according to the present invention is a DNA encoding a protein which has the amino acid sequence of SEQ ID NO: 2, wherein at least the glycine residue at position 56 is replaced by another amino acid residue .

 In addition, a more specific embodiment of the DNA of the present invention is a DNA encoding a protein which has the amino acid sequence of SEQ ID NO: 2 and which includes any of the following mutations: (i) replacement glycine residue at position 56 of SEQ ID NO: 2 by another amino acid residue; (ii) replacing the glycine residue at position 56 of SEQ ID NO: 2 with another amino acid residue, and replacing the alanine residue at position 55 of SEQ ID NO: 2 with another amino acid residue, (iii ) replacing the glycine residue at position 56 of SEQ ID NO: 2 with another amino acid residue, and replacing the aspartic acid residue at position 137 of SEQ ID NO: 2 with another amino acid residue.

 Replacing the glycine residue with a serine residue is a specific example of replacement at the 56th position. Replacement of the alanine residue with a threonine residue is a specific example of replacement at 55th position. The replacement of the aspartic acid residue with a glycine residue is a specific example of replacement at the 137th position.

mentionnés précédemment sont les ADN appelés lysL562, lysE564 et lysE565 qui sont décrits ici dans les exemples qui suivent. Il s'agit de mutants de gènes isolés à partir de Brevibacterium lactofermentum sous forme d'homologues du gène lyse décrit pour les bactéries Corynebacterium. De ce fait, l'ADN selon la présente invention est appelé aussi "lysE mutant", et la protéine codée par l'ADN de la présente invention est appelée "LysE mutante". More specific embodiments of the DNA according to the present invention encoding proteins having the substitutions

mentioned above are the DNAs designated lysL562, lysE564 and lysE565 which are described here in the examples which follow. These are mutants of genes isolated from Brevibacterium lactofermentum as homologs of the lysis gene described for Corynebacterium bacteria. As a result, the DNA of the present invention is also referred to as "mutant lysE", and the protein encoded by the DNA of the present invention is referred to as "mutant LysE".

 As the DNA encoding the LysE protein of coryneform bacteria, the wild-type lysE nucleotide sequence of Brevibacterium lactofermentum is shown in SEQ ID NO: 1 and the

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 The amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

 It has been found that glycine at position 56 from the amino terminus is converted to serine in the amino acid sequence of the lysE564 encoded protein, relative to the amino acid sequence of SEQ ID NO: 2 encoded by wild-type lysE. It has been found that glycine at position 56 from the amino terminus is converted to serine and that aspartic acid at position 137 is converted to glycine in the amino acid sequence of the protein encoded by lysE562, relative to to the amino acid sequence of SEQ ID NO: 2 encoded by wild-type lysEde. It has also been found that glycine at position 56 from the amino terminus is converted to serine and that alanine at position 55 is converted to threonine in the amino acid sequence of the protein encoded by lysE565 relative to the amino acid sequence encoded by wild type lysE. The common mutation point was found to be the replacement of glycine by serine at position 56. It was determined that modification at this position was important, particularly for the production of a secretory carrier that has activity of secretion of L-lysine in a bacteria assimilating methanol.

 The DNA of the present invention can encode an amino acid sequence including a substitution, deletion, insertion or addition of one or more amino acid residues at positions other than positions 55, 56 and 137, provided that encoded mutant LysE has one of the mutations mentioned above and shows the function of the LysE protein in a bacteria assimilating methanol. The term "many" as used herein varies depending on the positions of the amino acid residues in the three-dimensional structure of the protein and the types of amino acids. However, it preferably means 2 to 10 amino acid residues, more preferably 2 to 5 and particularly preferably 2 to 3 amino acid residues.

 DNA encoding a protein substantially identical to the mutant LysE mentioned above can be obtained by modifying the nucleotide sequence of the mutant lysE. For example, it is possible to employ site-directed mutagenesis so that substitution, deletion, insertion or addition of one or more residues occurs.

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 of amino acids at a specific site. In addition, it is also possible to obtain a modified DNA as described above by means of known mutation treatments which include, for example, a method of treating the DNA before the in vitro mutation treatment with hydroxylamine, by for example, a method of treating a microorganism, for example an Escherichia bacterium, containing the DNA before the ultraviolet ray mutation treatment or a mutagenic agent used in a common mutation treatment such as N-methyl -N'-nitro-Nnitrosoguanidine (NTG) or nitrous acid. These methods are described by Sambrook, J., Fritsch, E.F., and Maniatis, T., in "Molecular Cloning A Laboratory Manual, Second Edition," Cold Spring Harbor Laboratory Press (1989).

 It is possible to obtain a DNA encoding a protein substantially identical to the mutant LysE by expressing DNA including any of the mutations previously mentioned in a methanol-assimilating bacterium and examining the activity of the expressed product.

 In the present invention, the positions of the amino acid residues are not necessarily absolute positions in each LysE protein, but positions relative to positions in the amino acid sequence of SEQ ID NO: 2. For example, when a residue of amino acid is deleted at the N-terminal end of position 56, in the amino acid sequence of SEQ ID NO: 2, the 56th amino acid residue becomes the 55th amino acid residue from the N-terminus. terminal. In this case, since the 55th amino acid residue from the N-terminus is an amino acid residue corresponding to the 56th amino acid residue in SEQ ID NO: 2, it will be called the 56th amino acid residue.

 The DNA encoding the LysE protein of coryneform bacteria or its homologous protein, i.e. the lysis gene or its homologous gene, can be obtained from any microorganism provided that the microorganism has gene variants that can express the secretory activity of L-lysine in a methanol-assimilating bacterium. Specifically, such microorganisms may be for example Coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium lactofermentum, Escherichia bacteria as

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 Escherichia coli, Pseudomonas bacteria such as Pseudomonas aeruginosa, Mycobacterium bacteria such as Mycobacterium tuberculosis.

 Examples of lysE homologous genes include DNA encoding a protein that can be hybridized to a probe having the nucleotide sequence of SEQ ID NO: 1 or a portion thereof under stringent conditions, and that encodes a protein that exhibits the LysE protein function in a methanol-assimilating bacterium as a result of the above-mentioned amino acid substitution. The aforementioned stringent conditions include conditions in which a specific so-called hybrid is formed, and a non-specific hybrid is not formed. It is difficult to express this condition clearly by using any numerical value. However, for example, stringent conditions include conditions in which DNAs having a high homology, for example DNAs having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, are hybridized to each other, while DNAs having a homology less than the above homology do not hybridize with each other. Alternatively, the stringent conditions are illustrated by conditions in which DNAs hybridize to each other at a concentration of salts corresponding to ordinary washing conditions in Southern hybridization, i.e. x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS, at 60 ° C.

 It is also possible to use as a probe a partial sequence of the nucleotide sequence of SEQ ID NO: 1. It is possible to produce probes by PCR using, as primers, oligonucleotides based on the nucleotide sequence of SEQ ID NO: 1, and as a template a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1. When a DNA fragment of about 300 bp is used as the probe, the washing conditions for hybridization can be, for example, 2 × SSC. 0.1% SDS at 50 ° C.

 To improve the expression of the mutant lysE gene in a methanol-assimilating bacterium such as a Methylophilus bacterium or a Methylobacillus bacterium, it is possible to ligate the gene fragment containing the mutant lysE gene to a vector that is functional in a methanol-assimilating bacterium. , preferably a copy type vector

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 multiple, to prepare a recombinant DNA that is then used to transform a host like a bacteria assimilating methanol. Alternatively, it is possible to incorporate the mutant lysis gene into a transposon that is introduced into the chromosome. In addition, it is possible to ligate upstream of the mutant lysE gene a promoter that induces active transcription in a bacteria assimilating methanol.

 WO 97/23597 describes lysE and shows only the lysE gene of coryneform bacteria introduced into a coryneform bacterium. In addition, this document mentions only L-lysine as a secreted amino acid, and describes a protein secretion system including LysE having a structure containing 6 transmembrane helices. However, the Applicant has confirmed that LysE derived from coryneform bacteria is not functional in bacteria assimilating methanol.

In addition, the factor obtained is an L-lysine secretion support which includes a substitution mutation of an amino acid at a specific site, and it is not possible to deduce such a factor from the previous patents which concern lysis
The methanol-assimilating bacterium according to the present invention is a methanol-assimilating bacterium in which the above-mentioned DNA according to the present invention is introduced in a form which can be expressed and which is capable of producing L-lysine or L-Lysine. arginine. The methanol-assimilating bacterium according to the present invention can be obtained by introducing the DNA of the present invention into a methanol-assimilating bacterium having the ability to produce L-lysine or L-arginine. The methanol-assimilating bacterium according to the present invention can also be obtained by conferring the ability to produce L-lysine or L-arginine on a methanol-assimilating bacterium into which the DNA of the present invention has been introduced. In addition, the bacteria assimilating methanol according to the present invention may be a bacterium to which the ability to produce L-lysine or L-arginine has been conferred by introducing the DNA of the present invention in a form which can to be expressed.

 The Methylophilus bacteria and the Methylobacillus bacteria mentioned above are examples of bacteria assimilating methanol.

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 It is possible to obtain a bacterium that assimilates methanol which is capable of producing L-lysine or L-arginine by conferring an ability to produce L-lysine or L-arginine on a wild-type strain. a bacterium assimilating methanol. It is possible to use methods conventionally used to culture coryneform bacteria or Escherichia bacteria to confer the ability to produce L-lysine or L-arginine. For example, such methods include, but are not limited to, obtaining auxotrophic mutant strains, analog-resistant strains, or mutant strains for metabolic regulation, creating recombinant strains in which an enzyme of the biosynthetic system of the L-lysine or L-arginine is activated (see "Amino Acid Fermentation", Japan Scientific Societies Press [Gakkai Shuppan Center], 1st edition, published May 30, 1986, pp. 77-100). It is possible to individually confer properties of auxotrophy, resistance to an analogue, mutation concerning metabolic regulation, in particular, individually or in combination, when culturing bacteria producing L-lysine or L-arginine. . The enzyme of the biosynthetic system can be activated individually or it is possible to activate two or more enzymes of this type in combination. In addition, it is possible to confer properties including auxotrophy, analogue resistance, metabolic regulation mutation and to confer in combination the stimulation of the enzyme of the biosynthetic system.

amino-e-caprolactame, l'a-aminolauryllactame, un analogue de l'acide aspartique, un médicament de type sulfonamide, un quinoïde ou la Nlauroylleucine. For example, L-lysine-producing bacteria may be cultured to be auxotrophic for L-homoserine or for L-threonine and L-methionine (Japanese Published Patent Application Nos. 48-28078 and 56). -6499) or to be auxotrophic for inositol or acetic acid (Japanese Patent Applications Laid-open Nos. 55-9784 and 56-8692), or to be resistant to oxalysine, lysine hydroxamate, S- (2-aminoethyl) cysteine, methyllysine, α-chlorocaprolactam, DL-α-

amino-ε-caprolactam, α-aminolauryllactam, an aspartic acid analog, a sulfonamide drug, a quinoid or Nlauroylleucine.

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 L-arginine-producing bacteria can be cultured to be resistant to a certain agent, for example a sulfonamide drug, 2-thiazolalanine, α-aminohydroxyvaleric acid, so that they are auxotrophic for L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine or L-tryptophan in addition to resistance to 2-thiazolalanine (Japanese patent application Public Notice 54-44096), to be resistant to ketomalonic acid, fluoromalonic acid or monofluoroacetic acid (Japanese Patent Application Laid-open No. 57-18989), to be resistant to argininol (Japanese Patent Application Laid-Open No. 62-24075), to be resistant to X-guanidine (X represents a fatty acid derivative or a chain aliphatic, Japanese patent application made available to the public n 2-186995), so that they are resistant to 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine, so that they are resistant to arginine hydroxamate and 2-thiouracil, to be resistant to arginine hydroxamate and 6-azauracil (Japanese Patent Application Laid-Open No. 57-150381), to be resistant to a histidine analog or a tryptophan analogue (Japanese Patent Application Laid-Open No. 52-114092), to be auxotrophic for at least one of methionine , histidine, threonine, proline, isoleucine, lysine, adenine, guanine and uracil (or a precursor of uracil) (Japanese Patent Application Laid-Open No. 52 -99289), so that they are resistant to arginine hydroxamate (Japanese Patent Application Laid-open No. 51-6754), for that they are auxotrophic for succinic acid or resistant to a nucleic acid base analogue (Japanese Patent Application Laid-Open No. 58-9692), so that they are unable to metabolize arginine and to be resistant to an arginine and canavanine antagonist and to be auxotrophic for lysine (Japanese Patent Application Laid-open No. 52-8729), so that they are resistant to arginine, arginine hydroxamate, homoarginine, D-arginine and canavanine, or that they are resistant to arginine hydroxamate and 6-azauracil (application

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 Japanese Patent Publication No. 53-143288), or to be resistant to canavanine (Japanese Patent Application Laid Open No. 53-3586).

 In the following, methods for imparting or improving the ability to produce L-amino acids by improving a gene of an L-amino acid biosynthesis enzyme are provided by way of example.

 It is possible to confer an ability to produce L-lysine for example by increasing the activities of dihydrodipicolinate synthase and aspartokinase. It is possible to improve the activities of dihydrodipicolinate synthase and aspartokinase in a methanol-assimilating bacterium by transforming the methanol-assimilating bacteria host into a recombinant DNA that has been prepared by ligating a coding gene fragment. dihydrodipicolinate synthase and a gene fragment encoding aspartokinase with a vector which is functional in the methanol-assimilating bacterium, preferably a multi-copy type vector. The activities of dihydrodipicolinate synthase and aspartokinase are enhanced as a result of the increased copy number of the genes encoding the enzymes in the transformed strain. In the following, dihydrodipicolinate synthase, aspartokinase and aspartokinase III are also called DDPS, AK and AKIII, respectively.

 Any microorganism can provide the genes that encode DDPS and AK provided that the selected microorganism contains genes that can express DDPS activity and AK activity in a methanol-assimilating bacterium. Such a microorganism may be a wild-type strain or a mutant strain derived from the previous one.

Specifically, such microorganisms may be for example the strain of E. coli (Escherichia coli) K-12, the strain of Methylophilus methylotrophus AS1 (NCIMB10515) and the strain of Methylobacillus glycogenes T-11 (NCIMB11375). It is possible to obtain these genes by PCR using primers synthesized on the basis of known nucleotide sequences of DDPS (dapA, Richaud, F. et al., J. Bacteriol., 297 (1986)) and AKIII (lysis, Cassan, M., Parsot, C. Cohen, GN and Patte, JC, J. Biol Chem., 261, 1052 (1986)) and using as a template

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 the chromosomal DNA of a microorganism such as E. coli K-12. Specific examples include dapA and / or E. coli.

 Preferably, the DDPS and AK used in the present invention are not subject to feedback inhibition by L-lysine. Wild type DDPS from E. coli is known to be back-inhibited by L-lysine (see US Pat. Nos. 5,661,012 and 6,040,160) and wild-type AKIII from E. coli is subject. suppression and feedback inhibition by L-lysine. Therefore, dapA and lysC preferably encode DDPS and AKIII, respectively, each containing a mutation that eliminates feedback inhibition by L-lysine upon introduction into a methanol-assimilating bacterium. In the following, the DDPS which contains a mutation that eliminates feedback inhibition by L-lysine may also be referred to as "mutant DDPS" and the DNA encoding the mutant DDPS may also be referred to as "mutant dapA" or "dapA *". . AKIII derived from E. coli that contains a mutation that eliminates feedback inhibition by L-lysine may also be referred to as "mutant AKIII", and DNA encoding mutant AKIII may also be referred to as "mutant".

 However, it is not always necessary that DDPS and AK be mutated according to the present invention. It is known that, for example, DDPS derived from Corynebacterium bacteria is not back-inhibited by L-lysine (see published Korean Patent Application No. 92-8382, and US Patent Nos. 5,661,012 and 6,040,160).

 A wild-type dapA nucleotide sequence from E. coli is exemplified in SEQ ID NO: 3, and the amino acid sequence of the wild-type DDPS encoded by this nucleotide sequence is presented as a example in SEQ ID NO: 4.

 The DNA encoding the mutant DDPS which does not undergo feedback inhibition by L-lysine may be a DNA encoding the DDPS having the amino acid sequence of SEQ ID NO: 4, including the replacement of the histidine residue at position 118 of SEQ ID NO: 4 by a tyrosine residue.

In addition, the DNA encoding the mutant AKIII that does not undergo feedback inhibition by L-lysine may be a DNA encoding AKIII having an amino acid sequence including the replacement of threonine at position 352 by an isoleucine residue. (For the sequence of AKIII, see U.S. Patent Nos. 5,661,012 and 6,040,160).

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 The plasmid used for gene cloning can be any plasmid provided that it can replicate in microorganisms such as Escherichia bacteria. Specifically, such plasmids include, for example, pBR322, pTWV228, pMW119 and pUC19.

 Vectors that are functional in Methylophilus bacteria include, for example, a plasmid that can self-replicate in Methylophilus bacteria. Specifically, it may be RSF1010 which is a broad-spectrum host vector, and derivatives thereof, pAYC32 (Chistorerdov, A.Y., Tsygankov, Y. D.

Plasmid, 16, 161-167 (1986)), and pMFY42 (Gene, 44.53 (1990)), pRP301, pTB70 (Nature, 287, 396, (1980)).

 In addition, vectors that are functional in Methylobacillus bacteria include, for example, a plasmid that can autonomously replicate in Methylobacillus bacteria. It may be for example RSF1010 which is a broad-spectrum vector of hosts and derivatives thereof such as pMFY42 (Gene, 44,53, (1990)).

 To prepare a recombinant DNA by ligation of dapA and lysC to a vector that is functional in a methanol-assimilating bacterium, the vector is subjected to restriction enzyme digestion appropriate for the ends of a DNA fragment containing dapA and lysC. . Ligation is usually accomplished by means of a ligase such as T4 DNA ligase. The dapA and lysC genes can be incorporated in separate vectors or in the same vector.

 The broad-host RSFD80 plasmid (WO 95/16042) is known which can be used in the present invention as a plasmid having a mutant dapA encoding a mutant DDPS and a mutant lysC encoding a mutant AKIII. The E. coli strain JM109 transformed with this plasmid was named AJ12396 and was deposited with the National Institute of Bioscience of Advanced Industrial Science and Technology on October 28, 1993, and was assigned the FERM deposit number P-13936. Subsequently, it was the subject of an international deposit under the Budapest Treaty on 1 November 1994 and was assigned the FERM deposit number BP-4859. RSFD80 can be obtained from strain AJ12396 by a known method.

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 RSFD80 contains a mutant dapA where the wild-type dapA nucleotide sequence shown in SEQ ID NO: 3 is modified at position 623 of C at T. The histidine residue at position 118 of the wild-type DDPS of SEQ ID NO: 4, which is encoded by the wild-type dapA of SEQ ID NO: 3, is transformed into a tyrosine residue as a result of the above nucleotide change. In addition, RSFD80 contains a mutant lysC where the wild-type lysC nucleotide sequence is modified at position 1638 from C to T. This mutation leads to the mutant AKIII, but threonine at position 352 is transformed to isoleucine.

 Any method can be used to introduce the recombinant DNA prepared as described above into a Methylophilus bacterium or a Methylobacillus bacterium, provided that it leads to sufficient transformation efficiency. For example, electroporation can be used (Canadian Journal of Microbiology, 43, 197 (1997)).

 It is possible to achieve an improvement in the expression of a desired gene by introducing multiple copies of the gene onto the chromosomal DNA of a Methylophilus bacterium. It is possible to introduce multiple copies of dapA and lysC into the chromosomal DNA of a Methylophilus bacterium by homologous recombination. This can be achieved by targeting a sequence present on the chromosomal DNA in a number of multiple copies. It is possible to use as the sequence present on the chromosomal DNA in a number of multiple copies a repetitive DNA or an inverted repeat present at the end of a transposable element. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is possible to introduce multiple copies of the desired gene into chromosomal DNA by incorporating them into a transposon and then transferring it. In both of these methods, the activity of the desired genes will be enhanced as a result of the increased copy number of the desired genes in the transformed strains.

 In addition to the above gene amplification methods, it is possible to increase the expression of the desired gene by replacing an expression control sequence, such as dapA promoters and lysis, with stronger promoters (see Japanese Patent Application Laid Open No. 1-215280). Such strong promoters

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 may be for example the lac promoter, the trp promoter, the trc promoter, the tac promoter, the PR promoter or the phage lambda PL promoter, the tet promoter, the amyE promoter or the spac promoter.

The use of these promoters increases the expression of the desired gene, so that the activity of the desired gene product is amplified. It is possible to combine such methods of increasing gene expression with the gene amplification methods described above (increasing the number of copies of the desired gene).

 It is possible to prepare a recombinant DNA by ligating a gene fragment and a vector when the vector has been digested with a restriction enzyme corresponding to the end of the gene fragment.

Usually, ligation is performed with a ligase such as T4 DNA ligase. It is possible to use standard methods well known to those skilled in the art which include DNA ligation, chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation oligonucleotides used as primers.

Such methods are described by Sambrook, J., Fritsch, E. F., and Maniatis, T. in "Molecular Cloning A Laboratory Manual, Second Edition," Cold Spring Harbor Laboratory Press (1989).

 It is possible to improve not only the expression or the activity of the DDPS and AK genes but also other enzymes involved in the biosynthesis of L-lysine. Such enzymes include the diaminopimelate pathway enzymes such as dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase (see WO 96/40934 for all the above enzymes), phosphoenolpyruvate carboxylase (Japanese Patent Application Laid-Open) No. 60-87788), aspartate aminotransferase (Japanese Patent Application Laid-Open No. 6-102028), diaminopimelate epimerase and aspartic acid semialdehyde dehydrogenase, and enzymes of the aminoadipate pathway such as homoaconitate hydratase.

 Aspartokinase, aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase and diaminopimelate decarboxylase from Methylophilus methylotrophus are described in WO 00/61 723.

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 In addition, the microorganisms according to the present invention may have decreased activity of an enzyme that catalyzes a reaction to produce a compound other than L-lysine by a branching pathway of the L-lysine biosynthetic pathway. , or may be deficient in such an enzyme. Homoserine dehydrogenase (see WO 95/23864) is an example of enzymes that catalyze a reaction to form a compound other than L-lysine by a branching pathway of the Lysine biosynthetic pathway.

 It is also possible to use for L-arginine the techniques mentioned above to increase the activities of enzymes involved in the biosynthesis of L-lysine.

 It is possible to improve the production ability of L-arginine by increasing acetylornithine deacetylase activity, N-acetylglutamic acid-g-semialdehyde dehydrogenase activity, N-acetylglutamokinase activity and argininosuccinase activity (see Japanese Patent Application Laid-Open No. 5-23750).

 It is also possible to improve the production capacity of L-arginine by increasing the activity of glutamate dehydrogenase (EP 1 057 893 A1), argininosuccinate synthase (EPO 999 267 A1), carbamoylphosphate synthetase (EP 1 026 247 A1) or N-acetylglutamate synthase (see Japanese Patent Application Laid-Open No. 57-5693) or by destroying the gene encoding an arginine repressor (argR).

Production of L-lysine or L-arginine
It is possible to produce L-lysine or L-arginine by culturing a methanol-like bacterium such as a Methylophilus bacterium or a Methylobacillus bacterium, having an ability to produce L-lysine or L-arginine. It is possible to obtain L-lysine or L-arginine as described above from the medium by production and accumulation, and then to collect L-lysine or L-arginine from the culture.

 The microorganism used in the present invention can be cultured by a method typically used in the culture of microorganisms assimilating methanol. It is possible to use a medium

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 or a synthetic medium as a medium in the present invention, provided that it contains a carbon source, a nitrogen source, inorganic ions and other trace organic components.

 If methanol is used as the main carbon source, L-lysine or L-arginine can be produced at low cost. If used as the primary carbon source, methanol is added to medium at a concentration of 0.001 to 30%. Ammonium sulfate, for example, is added to the medium as a nitrogen source. It is also possible to add trace amounts of trace element such as potassium phosphate, sodium phosphate, magnesium sulphate, ferrous sulphate and manganese sulphate in small amounts.

 Usually, the culture is carried out under aerobic conditions by shaking or aeration while the pH is maintained between 5 and 9, and the temperature is maintained between 20 and 45 C, and the culture is typically completed in 24 to 120 hours. .

 It is possible to collect L-lysine or L-arginine from the culture by a combination of a process involving an ion exchange resin, a precipitation process and other known methods. .

 The present invention will be illustrated by the following non-limiting examples in which, unless otherwise indicated, reagents produced by Wako Pure Chemical Industries or Nakarai Tesque and media whose compositions are set forth below are used. For all media, the pH was adjusted with NaOH, KOH or HCl.

Medium LB: Bactotryptone (Difco) 10 g / L Yeast extract (Difco) 5 g / L NaCl 10 g / L pH 7.0
Steam sterilization was performed at 120 ° C. for 20 minutes.

LB agar medium: LB medium Bactogelose 15 g / L

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Steam sterilization was performed at 120 ° C. for 20 minutes.

Medium SEII: K2HPO4 1.9 g / L NaH2PO4 1.56 g / L MgSO4-7H2O 0.2 g / L (NH4) 2SO4 5 g / L CuSO4 # 5H2O 5 g / L MnSO4-5H2O 25 g / L ZnSO4 7H20 23 μg / L CaCl2 # 2H2O 72 mg / L FeCl3 # 6H2O 9.7 mg / L CaCO3 (Kanto Kagaku) 30 g / L Methanol 2% (v / v) pH 7.0
The different methanol components were steam sterilized at 121 ° C for 15 minutes and the methanol was added after cooling the medium sufficiently.

SEII agon medium: K2HPO4 1.9 g / L NaH2PO4 1.56 g / L MgSO4 # 7H2O 0.2 g / L (NH4) 2SO4 5 g / L CuSO4 # 5H2O 5 g / L MnSO4-5H2O 25 g / L ZnSO4 # 7H2O 23 g / L CaCl2 # 2H2O 72 mg / L FeCl3 # 6H2O 9.7 mg / L Methanol 0.5% (v / v) pH 7.0 Bactogelose (Difco) 15 g / L
The different methanol components were sterilized at 121 ° C for 15 minutes, and the methanol was added after cooling the medium sufficiently.

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Example 1 Construction of gene library / ysEmutant
First, the lysis gene, a homologous gene of the L-lysine secretion-increasing gene known for Corynebacterium bacteria, was cloned from a Brevibacterium bacterium, and the expression of the gene in a bacterium was attempted. Methylophilus.

(1) building pRSIysE
To introduce lysE into a Methylophilus bacterium, the known plasmid pRS (see International Patent Application in Japanese (Kohyo) No. 3-501682) was used to construct the plasmid pRSIysE for lysis expression. PRS is a plasmid having the of plasmid pVIC40 (published international patent application WO 90/04636, international patent application published in Japanese No. 3-501682) and obtained from pVIC40 by deletion of a DNA region encoding the threonine operon contained in the plasmid. Plasmid pVIC40 is derived from the broad-host plasmid vector pAYC32 (Chistorerov, AY, Tsygankov, YD, Plasmid, 1986,16, 161-167) which is a derivative of RSF1010.

 Specifically, pRS was constructed in the following manner.

Plasmid pVIC40 was digested with EcoRI, added to a solution of phenol / chloroform and stirred to terminate the reaction. The reaction mixture was then centrifuged, the top layer was collected and the DNAs were collected by ethanol precipitation and separated on a 0.8% agarose gel. A DNA fragment of about 8 kilobase bases (in the kpb sequence) containing the vector side was collected using the EASY TRAP Ver. DNA Collection Kit. 2 (Takara Shuzo). The vector region fragment of plasmid pVIC40 prepared as described above was autoligated using the DNA Ligation Kit Ver kit. 2 (Takara Shuzo). This ligation reaction solution was used to transform Escherichia coli (competent E. coli JM109 cells, Takara Shuzo). The cells were applied to LB agar medium containing 20 mg / L streptomycin and incubated overnight at 37 ° C. Each of the colonies which appeared on the agar medium was inoculated into LB liquid medium containing 20 mg / ml. Of streptomycin and cultured at 37 ° C. for 8 hours with stirring. Plasmid DNA was extracted

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 of each culture broth by the alkali-SDS method, and the structure of each plasmid was confirmed by digestion with restriction enzymes to obtain pRS.

 Then, plasmid pRStac containing the tac promoter was constructed from pRS according to the scheme of FIG. 1. The plasmid pRStac was constructed in the following manner. The pRS vector was digested with restriction enzymes EcoRI and PstI, and added to a solution of phenol / chloroform and mixed to complete the reaction. After centrifuging the reaction mixture, the top layer was collected and the DNAs were collected by ethanol precipitation, and separated on a 0.8% agarose gel. An 8 kbp DNA fragment was collected using the EASY TRAP Ver. DNA Collection Kit. 2 (Takara Shuzo). On the other hand, the PCR promoter region was amplified by PCR using as template the plasmid pKK223-3 (expression vector, Pharmacia) and the primers shown in SEQ ID NOS: and 8 (one 30-fold cycle consisting of denaturation at 94 ° C. for 20 seconds, hybridization at 55 ° C. for 30 seconds and an extension reaction at 72 ° C. for 60 seconds).

 Pyrobest DNA polymerase (Takara Shuzo) was used for the PCR. The DNA fragment containing the amplified tac promoter was purified using PCR prep (Promega) and then digested at previously established restriction enzyme sites in the primers, i.e. EcoRI and EcoT22I sites. Then, the reaction mixture was added to a phenol / chloroform solution and mixed to complete the reaction. After centrifuging the reaction mixture, the top layer was collected and the DNAs were collected by ethanol precipitation and separated on a 0.8% agarose gel. A DNA fragment of about 0.15 kbp was collected using EASY TRAP Ver. 2.

 The pRS vector digestion products and the tac promoter region fragment prepared as described above were ligated using the DNA Ligation Kit Ver kit. 2 (Takara Shuzo). This ligation reaction solution was used to transform Escherichia coli (competent E. coli JM109 cells, Takara Shuzo). The cells were plated on LB agar medium containing 20 mg / L streptomycin and incubated overnight at 37 C. Each of the colonies was inoculated.

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 appearing on the agar medium in LB liquid medium containing 20 mg / L of streptomycin and cultured at 37 ° C. for 8 h with stirring. Plasmid DNA was extracted from each culture broth by the alkali-SDS method and the structure of each plasmid was confirmed by digestion with restriction enzymes to obtain pRStac. PRStac was chosen as a plasmid in which the transcriptional directions of the streptomycin resistance gene on the pRS vector and the tac promoter were identical to each other.

 PRStac obtained as described above was digested with Sse8387I (Takara Shuzo) and SapI (New England Biolabs), added to a solution of phenol / chloroform and mixed to complete the reaction. After centrifuging the reaction mixture, the top layer was collected and the DNAs were collected by ethanol precipitation, and separated on a 0.8% agarose gel to obtain a DNA fragment. about 9.0 kbp.

 The lysing gene fragment was also amplified by PCR using the chromosome extracted from the strain Brevibacterium lactofermentum 2256 (ATCC13869) and the primers shown in SEQ ID NOS: as template and (denaturation at 94 C for 20 s, hybridization to 55 C for 30 s and extension reaction at 72 C for 90 sec). Pyrobest DNA polymerase (Takara Shuzo) was used for the PCR.

At this stage, so that the expression of the lysE gene is possible in a Methylophilus bacterium, the primers have been designed so that the nucleotides 9-15 bp from the translation initiation codon of the lysis gene are replaced by a a sequence known to be functional in a Methylophilus bacterium (Wyborn, NR, Mills, J., Williamis, SG and Jones, CW, Eur J. Biochem., 240, 314-322 (1996)).

The fragment obtained was purified with PCR prep (Promega) and then digested with Sse8387I and SapI. The reaction mixture was added to a phenol / chloroform solution and mixed to complete the reaction.

After centrifuging the reaction mixture, the top layer was collected and the DNAs were collected by ethanol precipitation, and further collected from a 0.8% agarose gel.

 The digests of the pRStac vector and the lysed gene region fragment prepared as described above were ligated using the DNA Ligation Kit Ver Kit. 2 (Takara Shuzo). We used

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 this ligation reaction solution to transform Escherichia coli (E coli competent cells JM109, Takara Shuzo). The cells were plated on LB agar medium containing 20 mg / L streptomycin and incubated overnight at 37 ° C. Each of the colonies appearing on the agar medium was inoculated with LB liquid medium containing 20 mg / L. streptomycin and cultured at 37 ° C for 8 h with shaking. Plasmid DNA was extracted from each culture broth by the alkali-SDS method and the structure of each plasmid was confirmed by restriction enzyme digestion and nucleotide sequence determined to obtain pRSIysE (Figure 1). In pRSIysE, the lysE gene was positioned such that its transcriptional direction was the same as that of the tac promoter.

(2) Introduction of pRSIysE into a Methylophilus Bacterium
PRSIysE obtained as described above in the strain Methylophilus methylotrophus AS1 (NCIMB10515) was introduced by electroporation (Canadian Journal of Microbiology, 43, 197 (1997)). In addition, pRS was also introduced into the AS1 strain as a control, in the same way as for pRSIysE. Thousands of colonies were obtained per g of DNA using pRS as a control, while only a few colonies were obtained with pRSIysE.

 When the plasmids were extracted from transformant strains considered to contain pRSIysE and when their nucleotide sequences were examined, it was found that a spontaneous mutation had been introduced into a lysE coding region for all the plasmids examined, and in some cases there was introduction of a nonsense mutation by which a codon encoding an amino acid was replaced by a stop codon that terminated the translation. In addition, in the other plasmid, deletion of the lysis gene was observed. The lysE function carried by such plasmids was considered to be lost.

 As described above, the frequency of introduction of pRSIysE carrying the full-length lysis gene into Methylophilus methylotrophus was extremely low, and only plasmids having a mutant lysE gene containing a mutation that eliminated its function could be introduced. Taking into account these facts in combination, it has been estimated that the introduction of the lysE gene into Methylophilus methylotrophus

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 had a lethal effect. This indicates that the lysis gene can not function universally for the secretion of L-lysine in heterogeneous bacteria.

 The strain of Methylophilus methylotrophus AS1 containing pRSIysE containing a SEII box mutation containing 50 mg / L streptomycin was applied and cultured overnight at 37 ° C. The cells were then scraped from approximately 10 cm 2 of The surface of the medium was inoculated with SEII production medium (20 ml) containing 50 mg / L streptomycin, and cultured at 37 ° C. for 34 h with shaking. After the end of the culture, the cells were removed by centrifugation and the concentration of L-lysine in the culture supernatant was determined by means of an amino acid analyzer (Nihon Bunko high speed liquid chromatography). As a result, substantially no strain was obtained in which the secretion of L-lysine was increased despite the introduction of the mutant lysE gene.

 As described above, the introduction of the already known lysE gene, derived from Corynebacterium bacteria, into bacteria assimilating methanol leads to a lethal effect. In strains having a lysE gene introduced only in small numbers, the introduced lysE gene undergoes a mutation or a deficiency, so that it is not functional. Since the proteins responsible for the secretion of amino acids are functional only when they are incorporated into the cell membrane, the conditions concerning the proteins and the membrane, such as the lipid composition, must be mutually compatible. As a result, it has been considered difficult to express a membrane protein from a heterologous organism so that its function is maintained. Thus, it has been found that if a method of introducing artificial mutations is used to positively introduce a greater number of mutations than natural mutations, it would be possible to successfully obtain from such mutants a gene. mutant lysE capable of secreting L-lysine. Based on this concept, a mutant library was constructed using a method of introducing mutations using hydroxylamine.

 In addition, the Applicant has considered that if a mutant lysE gene introducing a methanol-assimilating bacteria was introduced.

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 secretion activity of L-lysine in a methanol-assimilating bacteria, it would be possible to increase the degree of resistance to AEC (S- (2-aminoethyl) cysteine), an analogue of L-lysine. On the basis of this concept, a screening system described in the following has been developed. First, the construction of the mutant lysE library will be described in detail.

(3) Mutation processing of plasmid pRSIysE
E.coli strain JM109 containing the wild-type lysE-type plasmid pRSIysE (available from Takara Shuzo) was inoculated into LB liquid medium containing 20 mg / L streptomycin and cultured at 37 ° C for 8 h with shaking. Each plasmid DNA was extracted from each culture broth by the alkali-SDS method.

 Then, a mutation was introduced into the plasmid pRSIysE prepared by an in vitro mutation process using hydroxylamine, that is to say that a solution was prepared and incubated at 75 ° C. for 2 h or 3 h. containing 250 mM potassium phosphate buffer adjusted to pH 6.0, 400 mM hydroxylamine solution adjusted to pH 6.0, in each case as final concentration, and 2 g of plasmid pRSIysE which was adjusted to 200 l with water. Then, plasmid pRSIysE was collected from this solution using the EASY TRAP Ver. DNA Collection Kit. 2 (Takara Shuzo). The plasmids reacted with hydroxylamine were mixed for 2 h and the plasmids reacted with hydroxylamine for 3 h to obtain an aggregate of pRSIysE plasmids in which mutations were introduced at different rates.

 The aggregation of mutant pRSIysE plasmids obtained as described above, in which mutations were considered to have been introduced at different positions, was transformed and Escherichia coli (competent cells of E. coli JM109, Takara Shuzo) were transformed. with this aggregate of plasmids, these cells were applied to LB agar medium containing 20 mg / L streptomycin and incubated overnight at 37 ° C to construct a library in Escherichia coli. All colonies which appeared on the agar medium were scraped off, inoculated with LB liquid medium containing 20 mg / L streptomycin and cultured at 37 ° C for 8 h with shaking. Plasmid DNA was extracted from each culture broth by the alkali-SDS method and used as a library of mutant pRSIysE plasmids.

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Introduction of the mutant plasmid pRSIysE into a bacterium Methylophilus and production of L-amino acids (1) Screening of the lysE library containing artificial mutation for functional type lysE
The mutant pRSIysE plasmid library obtained as described above was introduced into the strain Methylophilus methylotrophus AS1 (NCIMB10515) by electroporation (Canadian Journal of Microbiology, 43, 197 (1997)). As a control, wild-type pRSIysE was introduced into strain AS1. As a result, with pRSIysE used as a control, the result of the previous examination was reproduced, and only a few colonies per g of DNA were obtained. On the other hand, with the mutant pRSIysE plasmid library, several hundred to several thousand colonies could be obtained. Examinations have so far shown that in substantially all the colonies that appeared when wild type pRSIysE was introduced, lysis lost its function due to the introduction of a natural mutation. That is, the frequency of introduction of pRSIysE containing the full-length lysis gene into Methylophilus methylotrophus was extremely low, and it was possible to introduce only plasmids having a mutant lysE gene containing a mutation which eliminated his function. Considering these facts in combination, it was felt that the introduction of the lysE gene into Methylophilus methylotrophus should lead to a lethal effect. On the other hand, as much larger colonies than above appeared when the mutant pRSIysE plasmid library was introduced, mutation was introduced by treatment with hydroxylamine in a high yield. .

 As described above, the mutant lysE library could be obtained as a mutant pRSIysE plasmid library. Then, a screening system was developed to obtain functional type lysE, which expresses an extracellular secretion activity of L-lysine.

 The strain of Methylophilus methylotrophus AS1 containing the mutant pRSIysE plasmid library obtained as described above was suitably diluted so that several hundred colonies appeared per dish, applied to SEII agar medium containing 50 mg / L of streptomycin and 3 g / L L-threonine and incubated at 37 C for 48 h. Colonies replicas were formed

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 appeared on SEII agar medium containing 3 g / L of L-threonine and 0.5, 7 or 10 g / L of AEC. When colony replicates were formed on a dish that did not contain AEC, the numbers of colonies that appeared on the initial box and on the replicate box after replica formation were identical. However, when colonies were replicated on the AEC-containing box, the number of colonies that appeared on the replicate box decreased significantly compared to the original box. The colonies formed on the AEC-replicated dish at each concentration were transferred to a new SEII-agar medium containing AEC and cultured, and AEC resistance was confirmed on the basis of the observation that only one colony could be formed on the same medium. From such strains, 100 strains were randomly selected and further screened.

(2) Production of L-amino acids by a strain in which the mutant plasmid pRSIysE was introduced
Of the strains of Methylophilus methylotrophus AS1 containing mutant pRSIysE that were believed to contain a mutation and confer resistance to AEC on a host, 100 strains were applied to one SEII box containing 50 mg / L streptomycin and It was grown overnight at 37 ° C. The cells were then scraped off about 10 cm 2 from the surface of the medium, inoculated with SEII production medium (20 ml) containing 50 mg / L streptomycin. and cultured at 37 ° C. for 48 hours with stirring. After the end of the culture, the cells were removed by centrifugation, and the L-amino acids contained in the culture supernatant were isolated by thin layer chromatography and roughly quantified by a method using ninhydrin detection. As a result, it was found that L-lysine and L-arginine were secreted outside the cells at different concentrations. These 100 strains were roughly classified into 5 groups based on their configurations.

 A typical strain was selected from each of the 5 groups, and pRSIysE561, pRSIysE562, pRSIysE563, pRSIysE564 and pRSIysE565 were designated plasmids contained in these strains. These plasmids were purified and the nucleotide sequences of their regions analyzed.

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 lysis. As a result, pRSIysE562 and pRSIysE563, and pRSIysE561 and pRSIysE564 were found to have completely identical sequences.

Therefore, pRSlys562, pRSIysE564 and pRSIysE565 were then examined.

 The nucleotide sequences of the LysE protein coding regions were analyzed in pRSIysE562, pRSIysE564 and pRSIysE565. As a result, lysE564 was found to include a substitution at the 166th position of G (guanine) by A (adenine) in the wild-type lysing DNA sequence shown in SEQ ID NO: 1. As a result , Gly (glycine) was replaced by Ser (serine) at the 56th position of the wild-type lysE amino acid sequence shown in SEQ ID NO: 2. It was also found that lysE562 included G (guanine) substitutions by A (adenine) at 166th position, from G (guanine) by A (adenine) to the 410th position of the wild-type lysing DNA sequence shown in SEQ ID NO: 1. As a result, Gly ( glycine) was replaced by Ser (serine) at 56th position and Asp (aspartic acid) was replaced by Gly (glycine) at the 137th position of the wild-type lysE amino acid sequence shown in SEQ ID NO: 2 It was further found that lysE565 included replacements of G (guanine) by A (adenine) at 166th pos and G (guanine) with A (adenine) at the 163rd position of the wild-type lysE DNA sequence shown in SEQ ID NO: 1. As a result, Gly (glycine) was replaced by Ser (serine). at 56th position and Ala (alanine) was replaced by Thr (threonine) at the 55th position of the wild-type lysE amino acid sequence shown in SEQ ID NO: 2. When pRSlysE562, pRSIysE564 was introduced again and pRSIysE565 in AS1 strains, these plasmids could be introduced at substantially the same frequency as pRS. The strains in which the plasmids were introduced by the same method as that described above were grown, and the concentrations of L-lysine and L-arginine in culture supernatants were quantified using an amino acid analyzer. (high performance liquid chromatography, Nihon Bunko). The results of the measurements are presented in Table 1 below.

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Table 1

<Tb>
<tb> Bacterial <SEP> strain <SEP> Quantity <SEP> of <SEP> L-lysine <SEP> Quantity <SEP> of <SEP> L-arginine
<tb> produced <SEP> (g / L) <SEP> produced <SEP> (g / L)
<tb> AS1 / pRS <SEP> 0.01 <SEP><SEP> 0.010
<tb> AS1 / pRSlyseE562 <SEP> 0.19 <SEP> 0.210
<tb> AS1 / pRSlysE564 <SEP> 0.20 <SEP> 0.240
<tb> AS1 / pRSlysE565 <SEP> 0.13 <SEP> 0.150
<Tb>

From the previous results, it was found that pRSIysE562, pRSIysE564 and pRSIysE565 had activity to substantially increase the secretion of L-lysine and L-arginine. Moreover, even when pRSIysE562, pRSIysE564 and pRSIysE565 were reintroduced into the AS1 strains, the activity of increasing the secretion of L-lysine and L-arginine was maintained. Thus, it has been demonstrated that the mutation of the strain having acquired AEC resistance was not introduced into the host but into the plasmid.

 The common mutation introduced into pRSIysE562, pRSIysE564 and pRSIysE565 was a replacement of Gly (glycine) with Ser (serine) at the 56th position of the wild-type lysE amino acid sequence shown in SEQ ID NO: 2. AJ110086 strain of E. coli JM109 transformed with pRSIysE564 having only this common mutation. This strain was deposited with the National Institute of Advanced Industrial Science and Technology on 26 September 2002 as an international deposit under the Budapest Treaty and was assigned the FERM deposit number BP-8196.

 The lysE562 and lysE565 genes can be readily obtained from lysE564 by methods well known to those skilled in the art.

Specifically, the lysE562 gene can be obtained by incorporating a fragment of the lysE564 coding region obtained from pRSIysE564, for example into pSELECTIM-1, a vector for the introduction of mutations by site-directed mutagenesis produced by Promega, and introducing a mutation. by site-directed mutagenesis with Altered SitesTM, a kit for introducing mutations by site-directed mutagenesis produced by Promega, to replace A (adenine) with G (guanine) at the 410th position in SEQ ID NO: 1. In the above process, for example, it is possible

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 to use the synthetic oligonucleotide of SEQ ID NO: 5 as a primer. The 20th nucleotide of SEQ ID NO: 5 is subjected to nucleotide substitution in the wild-type lysE gene. Similarly, it is possible to obtain the lysE565 gene by replacing G (guanine) with A (adenine) at the 166th position in SEQ ID NO: 1. For the introduction of a mutation, it is possible, for example, to The synthetic oligonucleotide of SEQ ID NO: 6 is used. The 16th and 19th nucleotides of SEQ ID NO: 6 are subjected to nucleotide substitution in the wild-type lysE gene.

dans laquelle le gène lysE562, lysE564 ou IysL65 avait été introduit pour tenter d'améliorer encore le rendement. Example 2 Introduction of an L-lysine Biosynthetic Enzyme Gene and of the LysE562, LysE564 or LysE565 Gene into Methylophilus methylotrophus
Since extracellular secretion of L-lysine was found to be increased by the introduction of the lysE562, lysE564 or lysE565 gene, the biosynthesis of L-lysine in a strain was increased.

in which the lysE562, lysE564 or lysL65 gene was introduced in an attempt to further improve the yield.

(1) Construction of the pRSdapA plasmid containing the dapA * gene
A plasmid having a gene encoding dihydrodipicolinate synthase which did not undergo feedback inhibition by L-lysine (dapA *) was prepared as an enzyme gene of the L-lysine biosynthesis system.

 Sse8387I and XbaI pRStac prepared in Example 1 were digested and added to a phenol / chloroform solution, and mixed to complete the reaction. After centrifuging the reaction mixture, the top layer was collected and the DNAs were collected by ethanol precipitation, and separated on a 0.8% agarose gel to collect a DNA fragment. about 9 kbp.

 The dapA * gene fragment was amplified by PCR using as template the known plasmid RSFD80 (see WO90 / 16042) containing this gene and the primers shown in SEQ ID NOS: and 12 (denaturation at 94 C for 20 s, hybridization to 55 C for 30 s and extension reaction at 72 C for 60 s). Pyrobest DNA polymerase (Takara Shuzo) was used for the PCR. The dapA fragment was purified

<Desc / Clms Page number 33>

 resulting with PCR prep (Promega) and then digested with restriction enzymes Sse83871 and XbaI. The reaction mixture was added to a phenol / chloroform solution and mixed to complete the reaction.

After centrifuging the reaction mixture, the top layer was collected, and the DNAs were collected by ethanol precipitation, and separated on a 0.8% agarose gel to collect a DNA fragment. about 0.1 kbp.

 The digests of the pRStac vector and the dapA * gene region fragment prepared as described above were ligated using the DNA Ligation Kit Ver kit. 2 (Takara Shuzo). This ligation reaction solution was used to transform Escherichia coli (competent E. coli JM109 cells, Takara Shuzo). The cells were plated on LB agar medium containing 20 mg / L streptomycin and incubated overnight at 37 ° C. Each of the colonies appearing on the agar medium was inoculated with LB liquid medium containing 20 mg / L. streptomycin and cultured at 37 ° C for 8 h with shaking. Plasmid DNA was extracted from each culture broth using the alkali-SDS method and the structure of each plasmid was confirmed by restriction enzyme digestion, and the nucleotide sequence was determined to obtain the plasmid pRSdapA. In the plasmid pRSdapA, the dapA * gene was positioned such that its transcriptional direction was the same as that of the tac promoter.

gènes lysE:562, lysE:564 et IysE565 avec dapA*, on a construit les plasmides pRSIysE562, pRSIysE564 et pRSIysE565 en insérant le gène dapA*. On a digéré pRSIysE562, lysE564 et lysE565 préparés dans l'exemple 1 avec l'enzyme de restriction SapI et on les a munis d'extrémités franches en utilisant la trousse DNA Blunting Kit (Takara Shuzo). De plus, on a digéré le plasmide pRSdapA avec les enzymes de restriction EcoRI et SapI, et on a séparé un fragment d'environ 1 kpb ayant le promoteur tac et la région dapA* sur un gel d'agarose à 0,8 %, et on l'a recueilli en utilisant EASY TRAP Ver. 2 (Takara Shuzo). On a muni ce fragment d'extrémités franches comme décrit ci-dessus et on l'a ligaturé à (2) Construction of a plasmid containing the dapA * gene and any of the lysE562, lysE564 and lysE565 genes
To evaluate the effect of the combination of any of the

lysE genes: 562, lysE: 564 and IysE565 with dapA *, plasmids pRSIysE562, pRSIysE564 and pRSIysE565 were constructed by inserting the dapA * gene. PRSIysE562, lysE564 and lysE565 prepared in Example 1 were digested with SapI restriction enzyme and blunt-ended using the DNA Blunting Kit (Takara Shuzo) kit. In addition, plasmid pRSdapA was digested with the restriction enzymes EcoRI and SapI, and an approximately 1 kbp fragment having the tac promoter and the dapA * region was separated on a 0.8% agarose gel. and collected using EASY TRAP Ver. 2 (Takara Shuzo). This blunt ended fragment was provided as described above and ligated to

<Desc / Clms Page number 34>

 each of the digestion products of pRSIysE562, lysE564 and lysE565 mentioned above using the DNA Ligation Kit Ver. 2 (Takara Shuzo).

 Escherichia coli (E.coli JM109 competent cells, Takara Shuzo) were transformed with each of the aforementioned ligation reaction solutions, applied to LB agar medium containing 20 mg / L streptomycin and incubated at 37 ° C for 1 hour. a night. The colonies that appeared on the agar medium were inoculated into LB liquid medium containing 20 mg / L streptomycin and cultured at 37 ° C for 8 h with shaking. Plasmid DNA was extracted from each culture broth using the alkali-SDS method, and its structure was confirmed by restriction enzyme digestion and nucleotide sequence determination to obtain plasmids pRSIysE562dapA, pRSIysE564dapA and pRSIysE565dapA. In these plasmids, the genes were positioned such that the transcriptional directions of lysE562, lysE564 and lysE565 were opposite to that of dapA *.

 When each of the plasmids pRSIysE562dapA, pRSIysE564dapA and pRSIysE565dapA obtained by the aforementioned method in the strain Methylophilus methylotrophus AS1 (NCIMB10515) was electroporated, transformant strains were obtained with pRSIysE562dapA and pRSIysE564dapA, whereas obtained no transformant strain with pRSIysE565dapA. This may be due to the fact that plasmid stability decreased in strain AS1.

(3) Preparation of L-lysine using Methylophilus bacteria containing lysE562 or lysE564 and dapA *
Each of the AS1 strains in which pRSIysE562dapA or pRSIysE564dapA obtained as described above was applied or pRSIysEdapA as a control was introduced onto a SEII dish containing 20 mg / L streptomycin and cultured overnight at 37 ° C. and the cells were scraped off over 0.3 cm 2 of the surface of the medium, inoculated into SEII production medium (20 ml) containing 20 mg / L streptomycin and cultured at 37 ° C for 34 hrs. shaking. After the end of the culture, the cells were removed by centrifugation and the concentration of L-lysine contained in

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 the culture supernatant by means of an amino acid analyzer (high performance liquid chromatography, Nihon Bunko). The results are shown in Table 2 below. Strains in which pRSIysE562dapA or pRSIysE564dapA had been introduced had an enhanced L-lysine accumulation. L-lysine accumulation in media was significantly improved over that obtained with pRSIysE562 or pRSIysE564 only. Thus, it can be seen that the limitation of the secretion rate was eliminated, and that the enhancement effect of the dapA * gene was synergistically manifested.

Table 2

<Tb>
<tb> Bacterial <SEP> Strain <SEP> Quantity <SEP> of <SEP> L-Lysine <SEP> Produced <SEP> (g / L)
<tb> AS1 / pRS <SEP><SEP> 0.10 <SEP>
<tb> AS1 / pRSlysE562dapA <SEP> 1.42
<tb> AS1 / pRSlysE564dapA <SEP> 1.40
<Tb>
Example 3: Introduction of the gene / ys564 into Methylobacillus glycogenes and production of L-amino acids (1) Preparation of pRS-lysE564-Tc
It was decided to confirm that lysE562, lysE564 and lysE565, which contain / ysEmutant, are functional in Methylobacillus bacteria. For this purpose, lysE564 was chosen, and plasmid pRSIysE564, a plasmid for lysE564 expression, was modified. Plasmid pRSIysE564 contains a streptomycin resistance gene.

However, since Methylobacillus glycogenes is initially resistant to streptomycin, it is not possible to screen the plasmid pRSIysE. As a result, a modified plasmid was constructed by inserting a tetracycline resistance gene into the plasmid pRSIysE which could be used in Methylobacillus glycogenes.

 First, pRS-lysE564-Tc containing the tetracycline resistance gene was constructed from pRSIysE564. Plasmid pRS-lysE564 was digested with the restriction enzyme EcoRI and added to a phenol / chloroform solution, and mixed to complete the reaction. The reaction mixture was centrifuged, and the top layer was collected. The DNAs were collected by ethanol precipitation, and

<Desc / Clms Page number 36>

 their digested ends were blunt-ended using the DNA Blunting Kit (Takara Shuzo). The DNA fragments were separated on a 0.8% agarose gel, and a DNA fragment of about 9 kbp was collected using the EASY TRAP Ver. DNA Collection Kit. 2 (Takara Shuzo).

 In addition, the region of the tetracycline resistance gene was amplified by PCR using the plasmid pRK310 (Pansegrau et al., J. Mol Biol 239, 663-663 (1994)) as template and the primers shown in SEQ ID. NOS: and 14 (a cycle consisting of denaturation at 94 ° C. for 20 s, hybridization at 55 ° C. for 30 s and an extension reaction at 72 ° C. for 60 s) was carried out 30 times. Pyrobest DNA polymerase (Takara Shuzo) was used for PCR. The amplified DNA fragment containing the region of the tetracycline resistance gene was purified using PCR prep (Promega), then the DNAs were collected by ethanol precipitation, blunt-ended and phosphorylated them using the Takara BKL kit (Takara Shuzo), added to a phenol / chloroform solution and mixed to complete the reaction. After centrifuging the reaction mixture, the top layer was collected, and the DNAs were collected by ethanol precipitation, and separated on a 0.8% agarose gel. A 1.5 kbp DNA fragment was collected using EASY TRAP Ver. 2.

 The tetracycline resistance gel can also be obtained by a PCR method similar to that described above, using another plasmid, for example the plasmid pRK2, as template, in place of pRK310.

 The pRSIysE564 vector fragment prepared as described above and the DNA fragment containing the tetracycline resistance gene were ligated using the DNA Ligation Kit Ver. 2 (Takara Shuzo). Escherichia coli (competent cells of E. coli JM109, Takara Shuzo) were transformed with this ligation reaction solution, applied to LB agar medium containing 20 mg / L streptomycin and 15 mg / L tetracycline and cultured overnight at 37 C. The colonies that appeared on the agar medium were inoculated into LB liquid medium containing 20 mg / L streptomycin and 15 mg / L tetracycline and cultured at 37 C for 8 h. with stirring. We extracted

<Desc / Clms Page number 37>

 the plasmid DNA of each culture broth using the alkali-SDS method, and the structure of each plasmid was confirmed by digestion with restriction enzymes to obtain pRS-lysE564-Tc.

(2) Introduction of pRS-lysE564-Tc into a Methylobacillus bacterium and production of L-amino acids
PRS-lysE564-Tc obtained as described above in the strain Methylobacillus glycogenes NCIMB11375 was introduced by electroporation (Canadian Journal of Microbiology, 43, 197 (1997)). As a control, pRK310 was likewise introduced into the strain Methylobacillus glycogenes NCIMB11375. As a result, colonies containing pRS-lysE564-Tc were obtained at substantially the same frequency as that obtained with the pRK310 control.

 PRSIysET was constructed by inserting a region of the tetracycline resistance gene into the wild-type lysE-like plasmid pRSIysE in the same manner as for pRS-lysE564-Tc, and an attempt was made to introduce it into the strain Methylobacillus glycogenes. the same way. However, no strain containing it was obtained. This is the same phenomenon as observed in the case of the strain Methylophilus methylotrophus AS1, and it has been estimated that wild-type lysis was also non-functional in Methylobacillus bacteria.

 The strain Methylobacillus glycogenes NCIMB11375 containing pRS-lysE564-Tc was applied to a SEII dish containing 10 mg / L tetracycline and grown overnight at 30 C. The cells were then scraped from about 10 cm.sup.2 of the surface of the medium and inoculated into SEII production medium (20 ml) containing 10 mg / L tetracycline and cultured at 30 ° C. for 60 h with stirring. After the end of the culture, the cells were removed by centrifugation, and the concentration of L-lysine contained in the culture supernatant was quantified by means of an amino acid analyzer (high performance liquid chromatography, Nihon Bunko). . The results obtained are shown in Table 3 below.

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Table 3

<Tb>
<tb> Bacterial <SEP> Strain <SEP> Quantity <SEP> of <SEP> L-Lysine <SEP> Produced <SEP> (g / L)
<tb> NCIMB11375 / pRK310 <SEP> 0.10
<tb> NCIMB11375 / pRS-lysE564-Tc <SEP> 1.40
<Tb>
(3) Construction of pRS-lysE564-dapA-Tc
L-lysine was found to accumulate in a medium due to the introduction of the lysE564 gene into the Methylobacillus glycogenes strain. This was considered to be due to increased secretion of L-lysine.

 Thus, the effect of increasing the expression of a Lysine biosynthesis gene and the lysE564 gene in combination in Methylobacillus glycogenes was examined in the same manner as in Example 2.

 A plasmid was constructed by incorporating a tetracycline resistance gene into the plasmid pRSIysE564dapA prepared in Example 2 (2) by the same method as in Example 3 (1). The plasmid obtained pRS-lysE564-dapA-Tc was called.

également "NCIMB1 1375/pRS-lysE564-Tc") et la souche de Methylobacillus glycogenes dans laquelle le plasmide pRK310 a été introduit à titre de témoin (appelée également "NCIMB11375/pRK310"). (4) Introduction of the pRS-lysE564-dapA-Tc plasmid into Methylobacillus glycogenes and production of L-amino acids
PRS-lysE564-dapA-Tc obtained as described above in the strain Methylobacillus glycogenes NCIMB11375 was introduced by electroporation. The concentration of L-amino acids in the supernatants of the culture broths was examined for the resulting transformant strain (also referred to as "NCIMB11375 / pRSlysE564-dapA-Tc"), the strain of Methylobacillus glycogenes mentioned above, in which pRS-lysE564 -Tc has been introduced (called

also "NCIMB1 1375 / pRS-lysE564-Tc") and the strain of Methylobacillus glycogenes in which the plasmid pRK310 was introduced as a control (also called "NCIMB11375 / pRK310").

 Each transformant strain was cultured on a SEII dish containing 10 mg / L tetracycline for 2 days at 30 C. Then the cells were scraped off on 10 cm 2 of the surface of the medium and inoculated into medium. SEII (20 ml) containing 10 mg / L

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 of tetracycline and cultured at 30 C for 60 h with stirring.

After the end of the culture, the cells were removed from a portion of the culture broth by centrifugation, and the concentrations of L-amino acids contained in the culture supernatant were quantified using an amino acid analyzer.

milieu contenant la souche NCIMB11375/pRS-lysES64-dapA-Tc. L'accumulation de L-lysine dans le milieu contenant la souche NCIMB11375/pRS-lysE564-dapA-Tc était améliorée par rapport au cas où seul pRSIysE564T avait été introduit. On considère que la limitation du taux de sécrétion était éliminée du fait de l'introduction du gène lysE564, et que l'effet de stimulation du gène dapA* était manifesté de manière synergique. The results obtained are shown in Table 4 below. A marked amount of L-lysine has accumulated in the

medium containing the NCIMB11375 / pRS-lysES64-dapA-Tc strain. The accumulation of L-lysine in the medium containing the strain NCIMB11375 / pRS-lysE564-dapA-Tc was improved compared to the case where only pRSIysE564T had been introduced. It is considered that the limitation of the secretion rate was eliminated due to the introduction of the lysE564 gene, and that the stimulation effect of the dapA * gene was synergistically manifested.

Table 4

<Tb>
<tb> Bacterial <SEP> Strain <SEP><SEP> Concentration of <SEP> L-Lysine <SEP> in <SEP>
<tb> supernatant <SEP> of <SEP> culture <SEP> (g / L)
<tb> NCIMB11375 / pRK310 <SEP> 0.20
<tb> NCIMB11375 / pRSIysE564T <SEP> 1.40
<tb> NCIMB11375 / pRS-lysE564-dapA-Tc <SEP> 1.62
<Tb>

Enclosed sequence list legend: SEQ ID NO: 1: Wild type lysE nucleotide sequence SEQ ID NO: 2: Wild type LysE amino acid sequence SEQ ID NO: 3: SEQ wild-type dapA nucleotide sequence ID NO: 4: wild-type DDPS amino acid sequence SEQ ID NOS: and 6: for mutation by specific mutagenesis of lysE562 SEQ ID NOS: and 8: for amplification of tac promoter SEQ ID NOS: and 10: for cloning Lysing SEQ ID NOS: 11 and 12: primers for cloning dapA * SEQ ID NOS: and 14: primers for amplifying the region of the tetracycline resistance gene

Claims (8)

A DNA encoding a mutant of the LysE protein of a coryneform bacterium, or a homologous protein thereof, characterized in that said mutant is a protein selected from the group consisting of: A) a protein which has the amino acid sequence of SEQ ID NO: 2, wherein at least the glycine residue at position 56 is replaced by another amino acid residue, and B) a protein which has the amino acid sequence of SEQ ID NO: 2 where at least the glycine residue at position 56 is replaced by another amino acid residue, and one or more amino acid residues at different positions of the amino acid residue; 56th residue are substituted, deleted, inserted or added, and when said mutant is introduced into a bacterium assimilating methanol, said mutant confers resistance to an analog of L-lysine.  2. DNA according to claim 1, characterized in that said DNA is selected from the group consisting of A) a DNA which has the nucleotide sequence of SEQ ID NO: 1 where a mutation results in the replacement of at least the 56th glycine residue of the encoded protein with another amino acid residue; B) a DNA which is hybridizable to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, or a probe prepared from said nucleotide sequence, and when said DNA or said probe is introduced into a bacteria assimilating methanol, said DNA or said probe encodes a protein that confers resistance to an L-lysine analogue.  3. DNA according to claim 1, characterized in that said other amino acid residue is a lysine residue.  4. DNA according to any one of the preceding claims, characterized in that said analogue of L-lysine is S- (2-aminoethyl) cysteine.  5. DNA according to any one of the preceding claims, characterized in that said bacterium assimilating methanol is a bacterium belonging to the genus Methylophilus or Methylobacillus. <Desc / Clms Page number 41>  6. Bacterium belonging to the genus Methylophilus or Methylobacillus, characterized in that the DNA according to claim 1 has been introduced into this bacterium in a form which can be expressed and in that it is capable of producing L-lysine or L-arginine.  7. Process for producing L-lysine or L-arginine characterized in that it comprises the steps of A) culturing the bacterium according to claim 6 in a medium for producing and accumulating L-lysine or L-arginine in the culture, and B) recovery of L-lysine or L-arginine from the culture.  8. Process according to claim 7, characterized in that the medium contains methanol as the main carbon source.